Latent HIV-1 infection is believed to represent the principal obstacle to curative AIDS therapy, as it allows the infection to persist in the face of antiretroviral therapy. In light of the ongoing discussion whether the intrinsic stability of the latent reservoir is the only contributing factor to the long half-life of the reservoir, or whether low-level viral replication contributes to the measured stability by continuous replenishment of the latent reservoir, interference with latency establishment may be an alternative strategy to therapeutically perturb the stability of the latent HIV-1 reservoir in infected patients. To identify potential new targets for therapeutic interference with HIV-1 latency, we have developed a T cell based experimental system in which HIV-1 latency establishment and maintenance in a cell population can be followed over time and quantitatively assessed at the single cell level. We demonstrate that latency establishment is the result of transcriptionally silent viral integration. Using pharmacological inhibitors of DNA methylation or histone deacetylation, we find no indication that these processes, which have been shown important for latency maintenance, play a role for the establishment of latent infection events. Our finding that FK506 (Tacrolimus, Prograf.), an immuno-suppressive compound that inhibits NFAT activation by interference with calcineurin, abolishes latency establishment without affecting active infection, suggests that latency establishment could indeed serve as a novel drug target. Based on our assay, we have developed a cell-based multiplexing technique that allows us to perform high throughput drug screening for inhibitors of latency establishment using flow cytometry. The HTS assay uses fluorescence signatures (fluorescent barcoding) to allow for the simultaneous analysis of drug effects at various concentrations (quantitative HTS (qHTS));changes in fluorescent patterns and population cell densities are used to quantify specific drug effects and possible drug toxicities. In this application, the assay will be combined with a series of verification assays that define the concentration-dependent influence of the compounds on methylated, latent and active infection to gain comprehensive insight on the compound effect on all HIV-1 transcription states. Once transferred to our robotic platform, the qHTS assay will be used to screen 50,000 compounds for inhibitors of latency establishment. Hits will be subjected to a medicinal chemistry program towards the development of lead compounds with the mid-term goal to identify and develop inhibitors of HIV-1 latency establishment that have less systemic side effects than tacrolimus and are more likely to be used in a standard HIV-1 therapeutic regiment with the intention of viral eradication. PUBLIC HEALTH RELEVANCE: Latent HIV-1 infection is believed to represent the principal obstacle to curative AIDS therapy, as it allows the infection to persist in the face of antiretroviral therapy, however, previous attempts to eradicate the HIV-1 reservoirs by reactivating latent infection events failed. Here, we demonstrate that the establishment of a latent infection can be prevented and that latency establishment can serve as a novel drug target. In this application, we propose to perform drug screening for inhibitors of latency formation with the mid- to long-tem goal of developing drugs to be used in HIV-1 therapy with the intention of viral eradication.